As a method for performing measurement (qualitative or quantitative analysis) of a target component, an enzymic method using oxidation-reduction commonly is put in practice. According to this method, for example, an oxidizing substance is produced from a target component that is to be measured, this oxidizing substance and a color-developer that produces a color-developed compound through oxidation are caused to react by an oxidizing enzyme, and the light absorbance of the color development that occurs is measured. In this method, the light absorbance corresponds to the amount of color-developed compound produced, the amount of color-developed compound produced corresponds to the amount of oxidizing substance produced, and the amount of oxidizing substance corresponds to the amount of target component. That is to say, a target component can be measured indirectly through such oxidation-reduction, by detecting the color development that occurs (produced color-developed compound).
Examples of a color-developer used in the enzymic method include Trinder's reagent and N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt (product name DA-64; manufactured by Wako Pure Chemical Industries, Ltd.). Furthermore, known examples of a color-developer that produces a phenothiazine-derivative color, such as methylene blue, which is a color-developed compound, include 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (product name DA-67; manufactured by Wako Pure Chemical Industries, Ltd., hereinafter, also referred to as “DA-67”), 10-(acetylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(phenylcarbonyl)-3,7-bis(dimethylamino)phenothiazine as described in Patent Document 3, 10-(3-(methylcarboxyamino)-hexamethyl-amino)-phenothiazine as described in Patent Document 4, 10-(3-(methylcarboxyamino)-4-methyl-phenyl)-amino)-phenothiazine, 10-((3-(methylcarboxyamino methyl)-phenyl)-methylamino)-phenothiazine, 10-(1-naphthaleneamino)-phenothiazine, 10-(methyl)-phenothiazine, 10-(phenylamino)-phenothiazine, 10-(methylamino)-phenothiazine, and the like. Phenothiazine-derivative color has a maximum absorption at 590 to 670 nm, which is on a relatively long-wavelength side, and, thus, among these, a color-developer such as DA-67 that produces a phenothiazine-derivative color such as methylene blue is considered to be important because of the following reasons.
That is to say, a sample such as a bodily fluid contains not only a target component but also various other components, and these components may have an absorption in a range with a wavelength of around 500 nm or shorter. Thus, in the above-described enzymic method, in order to avert the influence of components other than the target component, a color-developed compound produced through oxidation-reduction preferably can be detected at a wavelength that is as long as possible. Thus, a color-developer such as DA-67 that produces a phenothiazine-derivative color having a maximum absorption at 590 to 670 nm as described above is considered to be particularly useful.
However, for example, samples containing hemoglobin (hereinafter, referred to as “Hb”), such as a whole blood sample or a blood cell sample, may exhibit an absorption that can affect detection of a color at approximately 570 to 630 nm. Moreover, when Hb is methylated, the samples may exhibit an absorption that can affect detection of a phenothiazine-derivative color at approximately 570 to 670 nm. Accordingly, even with the above-described color-developer that produces a phenothiazine-derivative color, the measurement may be influenced in the case where the color-developer is applied to these sort of samples.
Moreover, there are clinical chemistry-specific automatic analyzing apparatuses in which the detection wavelength is fixed, for example, not to 610 to 660 nm but to 700 nm. However, a phenothiazine-derivative color having a maximum absorption at 610 to 670 nm is extremely difficult to detect at 700 nm, and, thus, the measurement cannot be performed with present analyzing apparatuses having a detection wavelength at 700 nm.
For example, blue-colored methylene blue is reduced to colorless leucomethylene blue. Using these characteristics, a target component can be measured also by a method in which a phenothiazine-derivative color itself such as methylene blue is used as the color-developer, the phenothiazine-derivative color is reduced with a reducing substance produced by the target component, and the disappearance of blue color through such reduction (decrease in the phenothiazine-derivative color) is measured (Patent Documents 1 and 2). However, also in this case, problems as described above occur.    [Patent Document 1] JP 2000-214152A    [Patent Document 2] JP 2000-214155A    [Patent Document 3] JP H4-27839B    [Patent Document 4] JP S60-33479B